Analytica Chimica Acta 376 (1998) 83±91
Hapten design for compound-selective antibodies: ELISAS
for environmentally deleterious small molecules
Marvin H. Goodrow*, Bruce D. Hammock
Department of Entomology and Environmental Toxicology, University of California, One Shields Avenue, Davis, CA 95616, USA
Received 6 January 1998; accepted 11 June 1998
Abstract
We have developed an enzyme-linked immunosorbent assay (ELISA) for the herbicide simazine with virtually no recognition
of propazine and very low (8%) recognition of atrazine. In this research we have developed a generalized ``size-exclusion''
concept for designing immunogen hapten structures. The hapten should contain appendages smaller than those of the target
analyte thereby generating polyclonal antibodies that exclude recognition of analytes larger than the target molecule. The use
of this size-exclusion model, when extended to include coating/tracer haptens, is also able to predict suitable structures for
ELISA development. # 1998 Elsevier Science B.V. All rights reserved.
Keywords: Enzyme-linked immunosorbent assay (ELISA); Simazine; Atrazine; Propazine; s-Triazine herbicides; Diuron; Arylurea
herbicides; Hapten design
1. Introduction
The wide use of the s-triazine and arylurea compounds as pre- and post-emergent herbicides for the
control of broad-leaf weeds in agriculture, along highways and other rights-of-way, has distributed these
substances throughout our environment. This usage
[1,2] is detailed in Table 1. Note that whereas atrazine
is the major triazine herbicide consumed throughout
the USA, simazine has greater usage in California and
thus is of special interest to our regulatory agencies for
monitoring thousands of water and soil samples
annually [3]. The University of California, Davis,
located in the agricultural central valley, has a vested
*Corresponding author. Tel.: +1-530-752-6571; e-mail:
[email protected]
0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.
PII S0003-2670(98)00433-4
interest in clean soil and ground water. This is the
additional impetus to develop an immunoassay which
is selective for simazine in the presence of atrazine.
Since most enzyme-linked immunosorbent assays
(ELISAs) detect both atrazine and simazine, an
ELISA selective for simazine then would allow quantitation of both. Cyanazine, a moderately used herbicide in California, shows very low cross-reactivity in
our assays. It is currently being phased out of usage in
California, and thus is of less importance at this time.
2. Immunogen hapten design
The principal objectives of this research are to
develop ELISAs that selectively quantify one chemical compound in the presence of others in its class (i.e.
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M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
Table 1
Herbicide usage in the USA and California (kg10ÿ6)
USA [1]
California [2]
Triazines
Atrazine
Cyanazine
Prometryn
Propazine
Simazine
32.9
14.6
0.6
NAa
1.8
0.02
0.29
0.10
0
0.38
Arylureas
Diuron
Linuron
1.8
0.9
0.49
NAa
a
Not available.
analyzing for simazine in the presence of other
s-triazines) and to meet the demands of regulatory
agencies for inexpensive, reasonably accurate and
precise assessment of pesticide levels in the environment. Our efforts have been directed towards developing immunoassays for the most commonly used
herbicides, simazine and atrazine (Fig. 1), with a
secondary concentration on the arylureas, monuron
(VI) and diuron (VII). In-depth presentations of these
hapten design strategies may be found in our review
articles [4±6].
Since selectivity among a class is dependent on the
immunogen, structural design of the hapten becomes
increasingly important as the size of the target molecule becomes smaller and/or the number of determi-
Fig. 1. Chemical structures of some s-triazine and arylurea herbicides. Roman numerals are used in the text to refer to the compound.
Chemical abstract numbers are given below each structure.
M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
85
Fig. 2. Relative triazine cross-reactivities using a 6-position handle immunogen. All rabbit polyclonal antibodies were raised to the structure
shown in the inset with variations on the R group at the 4-amino position (using the convention of position 2 on the triazine having the
chlorine). The bars represent the cross-reactivites to simazine, atrazine and propazine assigning atrazine 100% cross-reactivity. Roman
numerals indicate structure number.
nant groups becomes fewer. Contrary to many immunogen hapten designs, we purposely avoid the use of
any functional group on the target molecule for conjugation to proteins. For example, the use of succinic
anhydride on an amino-substituted target molecule
converts a basic moiety into a neutral amide function,
introducing a strong hydrogen-bonding determinant,
vastly dissimilar to the native amino group. Such
hapten coupling also masks a key determinant group.
Finally in small molecules, the amide would be
immediately adjacent to the other determinant groups
to which one wishes to make antibodies, thus misdirecting antibody production. The following criteria
have governed our structure design for immunizing
haptens for polyclonal (PAbs) and/or monoclonal
(MAbs) antibody production:
1. The hapten should be a near perfect mimic of the
target structure in size, shape (geometry) and
electronic properties.
2. The chemistry of the target molecule must be
understood, reviewed and evaluated for methods
of insertion of the attachment handled by established chemistry.
3. The hapten handle (required for connecting the
target-mimic to a carrier protein) should in itself
not elicit antibody recognition. Thus, an innocuous
methylene handle often is most appropriate, the
length of which should be evaluated using one or
more homologs.
4. An appropriate functional group for covalent
attachment of the handle to the protein must be
compatible with the chemistry of the functional
groups on the target.
5. Several immunizing carrier proteins should be
evaluated (here we have found that keyhole
limpet hemocyanin (KLH), Limulus polyphemus
hemocyanin (LPH), and thyroglobulin conjugates
often produce the best PAbs). However, we
sometimes also try others selected from conalbumin (CONA), ovalbumin (OVA) and bovine
serum albumin (BSA); these may also serve as
coating antigens in the assay development
process.
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M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
To date, most ELISAs for atrazine (II) are actually
propazine (III) assays in that the antibodies (Abs)
recognize propazine better than atrazine. This was
indeed the case in our early assays (Fig. 2) where we
compared the cross-reactivities of rabbit polyclonal
antibodies (PAbs) produced from our immunogen
haptens (VIII and IX), having the 6-position handle
[7,8]. When the immunogen appendage was isopropylamino (VIII), propazine was recognized 2.5 times
better than atrazine and there was a negligible (0.1
times) Ab recognition of the smaller simazine molecule. When the immunogen appendage was the smaller ethylamino group (IX), the PAbs now provided
what might be called an atrazine (100% cross-reactivity (CR)) assay, but with increased recognition of
simazine (40% CR) and accompanying diminished
recognition of propazine (72% CR). Our subsequent
study using an even smaller appendage, a methylamino group (Fig. 3, X), generated a true simazine
(100% CR) immunoassay with lesser but still signi®cant recognition for atrazine (75% CR) and low (13%
CR) recognition for propazine [9].
We hypothesized that even more selective Abs
would be produced if we presented the unique and
Fig. 3. The structures of haptens used as immunizing and coating
antigens in a simazine immunoassay [9].
Fig. 4. Relative triazine cross-reactivities using a 2-position handle immunogen. All rabbit polyclonal antibodies were raised to the structure
shown as the inset with variations on the R group at the 4- and 6-amino positions (using the convention of position 2 on the triazing having the
chlorine). The bars represent the cross-reactivites to simazine, atrazine and propazine assigning atrazine 100% cross-reactivity. Roman
numerals indicate compound number.
M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
dissimilar features of simazine and atrazine structures
distal to the connecting handle. Thus we were faced
with the necessity of selecting a chlorine mimic capable of accommodating two appendages ± the target
structure and the handle. Our selection of sulfur as this
connecting link is reviewed in an earlier publication
[7]. It should also be noted that many 2-methylthiotriazines are commercial herbicides with physical and
biological properties very similar to the 2-chlorotriazine herbicides. Thus, in our initial studies, we concentrated on immunizing hapten structures which
presented these differences, ethylamino±isopropylamino (Fig. 4, XI) and the ethylamino±ethylamino
(XII) moieties, distal to the common 2-mercaptopropanoic acid handle for polyclonal antibody production
in rabbits. The ELISA developed from replacing the
isopropylamino group with a second ethylamino
group, resulted in a substantial increase in simazine
recognition (about 15-fold) and a marked decrease (by
one-half) in the recognition of the larger propazine
molecule. A similar trend was also observed by Lawruk [10] using comparable haptens. In our most recent
studies [11,12] we found that reducing the size of one
appendage even more, from bis(ethylamino) (Fig. 4,
XII) to methylamino±ethylamino appendages (XIII),
produced antibodies with an astoundingly greater
recognition for the smaller simazine molecule (12
times that of atrazine, and 1200 times that of propazine, Fig. 5). This ``size-exclusion'' effect was even
more dramatic than what was observed on reducing
the size of the alkylamino in the series of structures
with the handle in the 6-position. Apparently antibody
pockets made to the small methylamino substituent
precluded any accommodation of the two isopropylamino groups of the propazine molecules.
Such an astounding effect, of course, necessitates
the obvious: preparation and evaluation of the
bis(methylamino)±mecaptopropanoic acid triazine
XIV as the immunogen hapten. This study is currently
in progress.
Concurrent with our studies, however, Lawruk et al.
[10] at the former Ohmicron laboratory, have also
developed a simazine-selective immunoassay. Using
PAbs derived from an immunogen with an aminobutanoic acid handle (shorter than ours, Fig. 2, IX)
and with a 6-[4,6-bis(ethylamino)±1,3,5-triazine-2yl]mercaptohexanoic acid hapten for the enzyme conjugate, they developed an excellent ELISA for sima-
87
Fig. 5. The structures of haptens used in immunizing and coating
antigens in an immunoassay of increased selectivity for simazine
[12].
zine. The assay showed an IC50 of 15 mg lÿ1, a limit of
detection of 0.07 mg lÿ1, and recognition of atrazine
and propazine of <1%.
3. Coating/tracer hapten design
In addition to our goal of designing haptens with
unique compound-selectivity among closely related
structures, we have synthesized numerous analogs and
homologs of the target structures for evaluation as
coating/tracer haptens. Our objective was to screen
multiple haptens searching for those providing ELISAs with the lowest levels of quanti®cation (LLQ),
often referred to erroneously as ``sensitivity''. (Since
the analytical chemist de®nes ``sensitivity'' as ``the
slope of the titration curve at the IC50 point'', we shall
avoid this term and focus on the amount of analyte
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M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
Fig. 6. Two ELISA formats commonly used in pesticide analysis. HRP ± horse radish peroxidase, Ht ± hapten tracer, Hc ± coating hapten,
A ± analyte, and Ab ± antibody.
present at the LLQ.) The advantages of using heterogolous haptens for immunogens and coating/tracer
haptens have been reported earlier [13] and the concept is employed extensively in our laboratory for the
triazines [7,8] and the arylureas [14,15]. The use of
heterologous assay systems has produced lower IC50s
than homologous assays [8]. Variables such as the
position of the handle, length of handles, position of
target appendages, and size of appendages offer a
variety of modi®cations for heterologous systems.
The conceptual steps with the two equilibria
involved in two common ELISA formats (antigencoated plate and enzyme tracer) are represented in
Fig. 6. A schematic representation of the quasi-equilibria using the antigen-coated plate format is illustrated in Fig. 7. Assuming no analyte (A) is present,
only the coating hapten equilibrium, KCH, (variable by
changing hapten structure) is in operation between
antibody (Y) and coating hapten (H) and a maximum
signal from a tracer antigen when attached to the Y±H
is observed. On the addition of a minute quantity of
analyte (A), this equilibrium is displaced towards the
formation of antibody±analyte (Y±A). This dramatically reduces the Y±H amount and hence the tracer
signal. Thus, for a ®xed quantity of antibody, the
lowest LLQ (and lowest IC50) is observed when the
af®nity of the antibody for the analyte is greater than
the af®nity of the antibody for the plate-coating hapten
(KAKCH). Hence, with a ®xed KA for Y±A, one can
Fig. 7. Schematic representation of the quasi-equilibria using
heterologous haptens in a competitive ELISA format occurring on
a hapten (H)±protein coated plate. In the absence of analyte (A) the
antibody will bind to the hapten-coated plate with an equilibrium
constant of KCH. If the antibody affinity for the hapten is the same
as for the analyte, there will be a 1:1 displacement of the antibody
from the plate by the hapten as indicated by the constant KA,
However, if the hapten binds to the antibody significantly less well
than the analyte (in practice often about 3%), then a small amount
of analyte will displace a large amount of antibody from the plate
giving a large reduction in signal and a lower IC50.
shift the overall equilibria by selecting a different
coating hapten with a decreased af®nity for the antibody (thus varying KCH). Assuming no change in the
slope of the curve, this produces a lower IC50 (as well
as decreased LLQ) assay for the target analyte.
Table 2 lists the coating/tracer hapten design criteria
for planning heterologous hapten syntheses which are
M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
89
Table 2
Coating/tracer hapten design criteria
1. Heterology of hapten structure
(a) Position of handle
(b) Composition of handle
(c) Length of handle
(d) Conjugation chemistry
2. Partial structure of target molecule
3. Consideration of cross-reactivity of hapten molecules
(or derivatives)
4. Alteration of immunizing hapten's chemical composition
detailed in former reviews [7,8]. Thus, synthesizing
many haptens and screening for an optimum coating
antigen has led to a monoclonal antibody (MAb)based ELISA for triazines at 40 ng lÿ1, but it was
not totally selective for any one species among the
class [16].
The substantial use of diuron (VII) in California
(Table 1), and its prominence in Germany for replacing the banned triazine herbicides [17] has renewed
our interest in improving existing ELISAs and developing new immunoassay techniques for this herbicide.
The former assay development by Karu et al. [15] was
derived from an ideal hapten through an extension of
an existing carbon chain (Fig. 8, XV) by methylene
groups. The best coating antigen of the three evaluated
consisted of an isomer (Fig. 8, XVI) with the methylene handle attached at the internal nitrogen. The IC50
was satisfactory at 2 mg lÿ1 with a LLQ of 0.6 mg lÿ1.
Using our rationale for a lower antibody af®nity for
the coating hapten, we had found that by replacing the
oxygen of the monuron immunizing hapten structure
with a sulfur to make a thiourea coating hapten, we
obtained a usable assay for monuron with a very low
IC50 of 0.5 mg lÿ1 [14]. The sulfur, requiring more
space than oxygen, probably could not ®t well in the
urea-established antibody pockets (based on a sizeexclusion model). Additionally there would be a substantially lower af®nity for this coating hapten structure due to the loss of hydrogen-bonding between the
thiocarbonyl and the antibody.
Using polyclonal antibodies made to a `monuron'
hapten (Fig. 9, XVII) and a structurally similar
diuron-type thiourea-HRP tracer hapten (XVIII), Kramer et al. [17] developed a diuron ELISA with an IC50
of 0.1 mg lÿ1 and a linear range 0.02±1 mg lÿ1.
Employing a newly developed ¯ow injection immu-
Fig. 8. The structures of haptens used for immunizing and coating
antigens in a monoclonal antibody based immunoassay for diuron
[15].
noaf®nity analysis (FIIAA), and the same reagents,
Kramer et al. found an IC50 of 0.1 mg lÿ1 and a linear
range 0.02±0.5 mg lÿ1. Using the mouse MAbs that
Karu [15] produced from the diuron hapten (XV),
Kramer improved on the IC50 in the ELISA using the
thio-diuron hapten (XIX), (IC50 of 4 mg lÿ1 and LLQ
of 0.6 mg lÿ1) [18]. This was a substantial improvement on values obtained by Karu using the same MAb
481 and the oxygen analog of XVIII as a coating
antigen (IC50 ca. 230 mg lÿ1). These three successful
examples in which a slight modi®cation of immunizing hapten structures produced low mg lÿ1 assays,
suggests the size-exclusion phenomenon might be
applied to other similar classes of pesticides such as
the organophosphates, carbamates, or the thiocarbamates. This principle might also be extended to replacing the 4-chlorophenyl moiety contained in several
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M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
4. Conclusions
Using a ``size-exclusion'' model for hapten design
and synthesis, we have developed polyclonal antibodies that selectively recognize the smaller molecules
of a class. Use of this same paradigm to design
heterologous coating/tracer haptens has provided
immunoassays with lower IC50s and lower levels of
quanti®cation.
5. Nomenclature
ELISA
cross-reactivity
IC50
Fig. 9. The structures of haptens used for immunizing and coating
antigens in an ELISA and FIIAA for Diuron [17,18].
pesticide immunogen haptens with a 4-iodophenyl for
use as a coating/tracer hapten.
Another broad generalization uncovered in our
years of ELISA developments was the observation
from screening multiple haptens, that when the best
immunogen hapten contained a long methylene chain
handle, the best coating/tracer handle would be very
short. This was especially true for the simazine ELISA
studied by Wortberg [9] where the immunogen handle
on the triazine (Fig. 2, X) was a 6-aminohexanoic
acid. Coating haptens bearing aminoacetic to 6-aminohexanoic acid handles in the same position were
evaluated. The IC50 for the shortest handle was
0.1 mg lÿ1 , whereas the IC50s of the others were in
the 100±1000 mg lÿ1 range. Additionally, the other
alkylamino substituents, being either ethylamino or
isopropylamino, showed no marked difference in their
IC50s. Apparently this latter appendage only had to be
larger than methylamino group of the immunogen
hapten to be appropriately less recognized by the
antibodies; the handle had to be short in both series.
lowest level of
quantification
(LLQ)
selectivity
sensitivity
enzyme-linked immunosorbent
assay
the ratio of the IC50 of the target
analyte to the IC50 of the test
analyte, expressed as a percent
the equivalence point, or the
concentration of analyte that inhibits maximum binding of the
antibody to the antigen by 50%
the lowest concentration of analyte which can be quantitatively
distinguished from the background singal
the ability of antibodies to bind
preferentially to one chemical
structure in the presence of other
substances
ratio of the rate of change of
signal to a corresponding change
of analyte concentration
Acknowledgements
The authors wish to acknowledge the many colleagues and co-workers who, over the last 12 years,
contributed their efforts to the triazine and arylurea
herbicide ELISA developments. At the University of
California, Davis, Departments of Entomology and
Environmental Toxicology were: S. Gee, A. Harris, R.
Harrison, H. Kido, P. Kramer, Q. Li, A. Lucas, P.
Schneider, Y. Sugawara, M. Wortberg and at the
University of California, Berkeley: A. Karu, D.
Schmidt and M. Bigelow. Financial support for some
of this research and for this review was received from
M.H. Goodrow, B.D. Hammock / Analytica Chimica Acta 376 (1998) 83±91
NIEHS Superfund Basic Research Program ESO4699,
NIEHS Center for Environmental Health Sciences
ESO5707, US, EPA Center for Ecological Health
Research CR819658, and Cosmo Oil Company Ltd,
Japan.
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Marvin H. Goodrow is a Research Organic Chemist in the
Departments of Entomology and Environmental Toxicology,
University of California, One Shields Avenue, Davis, California
95616, USA and Co-Principal Investigator for the NIEHS Superfund Basic Research Program.
Bruce D. Hammock is a Professor in the Departments of
Entomology and Environmental Toxicology, University of California, One Shields Avenue, Davis, California 95616, USA and CoPrincipal Investigator for the NIEHS Superfund Basic Research
Program.